This project is aimed towards a more compete understanding of the assembly of envelope glycoprotein complexes of human cytomegalovirus (HCMV) that mediate attachment and entry into different types of cells in the body. HCMV is a ubiquitous pathogen that causes a wide range of severe diseases in individuals with undeveloped or compromised immune systems. Therapies are limited, and candidate vaccines have failed to elicit protective immunity. Recent advances have contributed to the view that the attachment/entry-mediating glycoproteins encoded by HCMV include those that provide universal functions for entry into all cell types, and those that provide niche-specific functins for entry into specific cell types. Specifically, glycoprotein H/L (gH/gL) is likely required for etry into all cells types, whereas a complex of gH/gL bound by the UL128-131 proteins is required for entry into specific cell types. There is evidence that candidate vaccines have failed in part because that have not included UL128-131 as targets. It remains unclear how the virus incorporates gH/gL with and without UL128-131 into the mature virion envelope. Preliminary evidence suggests that the mechanism may involve glycoprotein O (gO), which is highly variable among strains (up to 28% of amino acids). The function of gO remains a mystery largely because of the lack of comprehensive analyses that take into account the diversity among strains. Furthermore, it is clear that during replication in cell types for which entry is UL128-131 independent, HCMV adapts by mutational inactivation of UL128-131. Strains differ in how rapidly they acquire these mutations and this may help explain the diverse presentation of HCMV disease. The results of these studies will greatly advance to our understanding of how HCMV attaches to, and enters the wide range of cell types in the body linked to various pathologies, and will more clearly define the virion envelope molecules that represent important vaccine targets. Two broad goals are represented in this proposal. 1. To study the mechanism of how gO affects the assembly of gH/gL complexes. It is likely that gO binds to a fraction of gH/gL in the infected cell, and blocks interactions with UL128-131. This hypothesis will be tested using an adenovirus vector expression system to reconstruct gH/gL complexes outside the context of HCMV. 2. Comparative analysis of diverse gO isoforms. To study the functional significance of gO diversity, of 5 different isoforms of gO have been cloned into adenovirus vectors and will be compared in their interactions with gH/gL/UL128-131, as well as their potential roles as components of the virion envelope. Also, gO isoforms will be swapped between strains of HCMV to study the assembly of gH/gL complexes, and to address the role of gO in the adaptive mutation of UL128-131.